Glutathione-dependent redox status of frataxin-deficient cells in a yeast model of Friedreich’s ataxia

Laboratoire d'Ingénierie des Protéines et Contrôle Métabolique, Département de Biologie des Génomes, Institut Jacques Monod, UMR 7592, CNRS, Universités Paris 6 and 7, 2 Place Jussieu, Tour 43, 75251 Paris Cedex 05, France.
Human Molecular Genetics (Impact Factor: 6.39). 07/2008; 17(18):2790-802. DOI: 10.1093/hmg/ddn178
Source: PubMed

ABSTRACT Friedreich's ataxia is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulphur cluster defects and high sensitivity to oxidative stress. Glutathione is a major protective agent against oxidative damage and glutathione-related systems participate in maintaining the cellular thiol/disulfide status and the reduced environment of the cell. Here, we present the first detailed biochemical study of the glutathione-dependent redox status of wild-type and frataxin-deficient cells in a yeast model of the disease. There were five times less total glutathione (GSH+GSSG) in frataxin-deficient cells, imbalanced GSH/GSSG pools and higher glutathione peroxidase activity. The pentose phosphate pathway was stimulated in frataxin-deficient cells, glucose-6-phosphate dehydrogenase activity was three times higher than in wild-type cells and this was coupled to a defect in the NADPH/NADP(+) pool. Moreover, analysis of gene expression confirms the adaptative response of mutant cells to stress conditions and we bring evidence for a strong relation between the glutathione-dependent redox status of the cells and iron homeostasis. Dynamic studies show that intracellular glutathione levels reflect an adaptation of cells to iron stress conditions, and allow to distinguish constitutive stress observed in frataxin-deficient cells from the acute response of wild-type cells. In conclusion, our findings provide evidence for an impairment of glutathione homeostasis in a yeast model of Friedreich's ataxia and identify glutathione as a valuable indicator of the redox status of frataxin-deficient cells.

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Available from: Renata Santos, Sep 26, 2015
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    • "Total glutathione levels were determined using 20 µg proteins of crude mitochondrial pellet or cytosol from striatum and midbrain with a modified version of the recycling enzymatic assay based on the reduction of each GSSG molecule to give two GSH molecules [51]. Standard curves were obtained with various concentrations of GSSG and the values were thus expressed as GSSG equivalent. "
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    ABSTRACT: Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival in vivo is still under debate. We addressed this issue in the brain of PARK2-/- mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2-/- brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen in vivo from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2-/- mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2-/- mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2-/- mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be elucidated, as complex I defect and prominent oxidative damage were ruled out.
    PLoS ONE 06/2014; 9(6):e99898. DOI:10.1371/journal.pone.0099898 · 3.23 Impact Factor
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    • "RT-qPCR analysis was performed exactly as described previously (21,34). Primer pairs used were: 5′-TCGCGTAAGGAACAGTTAGACA-3′ and 5′-GCTCTGCAGTGGCTTTCTTC-3′ for the APN1 gene; 5′-TAAGGGGCTTTGGACAGAAA-3′ and 5′-ATCATCCGGTGGCAAGAA-3′ for the NTG1 gene and 5′-CCAGGGGTAAGAGGTGTGATT-3′ and 5′-TCCAATTGATGAAATTTGGAAGA-3′ for the NTG2 gene or were described previously for the other genes (21,34). Relative expression was calculated using the equation described (53). "
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    ABSTRACT: Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15-30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H(2)O(2). On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.
    Human Molecular Genetics 06/2012; 21(18):4060-72. DOI:10.1093/hmg/dds230 · 6.39 Impact Factor
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    • "Glutathione is a major protective antioxidant in yeast cells. We have shown that Dyfh1 cells have low total glutathione content and a reduced GSH/GSSG ratio [8]. Since the double Dyfh1Dyca1 mutant was more resistant to H 2 O 2 , we tested if glutathione antioxidant defense was altered. "
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    ABSTRACT: Friedreich ataxia is the most common recessive neurodegenerative disease and is caused by reduced expression of mitochondrial frataxin. Frataxin depletion causes impairment in iron-sulfur cluster and heme biosynthesis, disruption of iron homeostasis and hypersensitivity to oxidants. Currently no pharmacological treatment blocks disease progression, although antioxidant therapies proved to benefit patients. We show that sensitivity of yeast frataxin-deficient cells to hydrogen peroxide is partially mediated by the metacaspase. Metacaspase deletion in frataxin-deficient cells results in recovery of antioxidant capacity and heme synthesis. In addition, our results suggest that metacaspase is associated with mitochondrial respiration, intracellular redox control and genomic stability.
    FEBS letters 12/2011; 586(2):143-8. DOI:10.1016/j.febslet.2011.12.002 · 3.17 Impact Factor
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